The enzyme acyl Coenzyme A-cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen databases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full length cDNA clones for these ACAT Related Gene Products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the Coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.
The intracellular formation of sterol esters from fatty acid and sterol is mediated by acyl-CoA cholesterol acyl transferase (ACAT). The pathological accumulation of cholesterol esters in atherosclerotic lesions has lead to intense pursuit of ACAT inhibitors as pharmacological agents. Microsomal ACAT preparations from various tissues display differential sensitivities to some of these agents including histidine (1) modifiers (2). This suggests that more than one protein mediates the esterification reaction, such as occurs in yeast (reviewed in FIG. 3]. Saccharomyces cerevisiae (budding yeast) has two ACAT related enzymes, Are1 and Are2, which are derived from separate genes and have been shown to independently esterify sterols (4,5). In terms of contribution to the sterol ester mass of the cell, Are1 is the minor isoform relative to Are2. These genes were identified based on sequence conservation to a human gene, ACAT1, which encodes an ACAT enzyme with homologs in many mammalian species . The human ACAT1 gene encodes a 550-amino acid polypeptide and is expressed in most tissues, predominantly placenta, lung, kidney, and pancreas (6). ACAT1 has been predicted to have two transmembrane domains (6) and has been immunolocalized to the endoplasmic reticulum (8,9). When murine ACAT1 was disrupted in induced mutant mice, homozygotes for the deletion were found to essentially lack ACAT activity in embryonic fibroblasts and have negligible amounts of cholesterol ester in the adrenal cortex and peritoneal macrophages (10). However, cholesterol ester accumulation was normal in hepatocytes while dietary cholesterol absorption, an indirect marker for intestinal cholesterol esterification, was indistinguishable from control litter mates. This is consistent with the concept of a multigene family for this activity.
ACAT isoenzymes may be required to perform the variety of physiological roles mediated by cholesterol esterification. Increases in cellular free cholesterol above certain levels are cytotoxic and are ameliorated by cholesterol ester formation (11). In hepatocytes, the bulk of cholesterol secreted in very low density lipoprotein (VLDL) is esterified intracellularly and determines apolipoprotein B secretion rates. Cholesterol esterification in the enterocyte may be necessary for cholesterol absorption from the lumen and secretion in chylomicrons into the lymph (15). The formation of cholesterol ester stores could also provide a readily available substrate for steroid hormone synthesis in steroidogenic tissues (16, 17). It is likely that different ACAT isozymes mediate each of these processes, and the data presented here support that hypothesis.
We reasoned that additional human ACAT proteins would have sequence similarity to regions conserved between human ACAT1 and yeast Are1 and Are2 (4). Accordingly, an ACAT consensus sequence was used to screen the data base of expressed sequence tags (dbEST). Several cDNA entries were identified which were transcribed from two independent human genes. This study is a description of the isolation of full-length cDNA clones for two ACAT related gene products (ARGP1 and ARGP2), examination of their pattern of tissue expression, and assays of enzymatic activity. We show that ARGP2 can catalyze the formation of sterol ester from cholesterol and oleoyl-CoA, leading us to rename this gene, ACAT2. By contrast, ARGP1 did not detectably esterify cholesterol and we propose that it performs acyl-CoA dependent acylation of other molecules, such as diacylglycerol.